N-methyl-D-aspartate receptors (NMDARs) are important for the normal function of the nervous system, but their excessive activation contributes to a number of developmental disorders and neurological diseases. This dual role imposes important constraints on possible therapeutic strategies aimed at ameliorating mental retardation and neurodegenerative disorders. Inhibition of NMDAR overactivity must be accomplished without interference of normal function. Our group was the first to show that the uncompetitive, open-channel blocker, Memantine, can curtail excessive activity of the NMDAR while leaving normal neurotransmission essentially unabated. Based in part on our work, Memantine was recently approved for clinical use in the USA. Here we develop dual-functional derivatives, the NO-Memantines, that use the Memantine moiety to target NO to S-nitrosylation site(s) on the NMDAR to further downregulate excessive activity better than Memantine alone. In our preliminary results, we have shown: (i) the glutamine/arginine/asparagine (Q/R/N) sites in the second transmembrane (M2) domains of NR1 and NR2 subunits form a specific binding site for Memantine; the binding of Memantine to this site can be used for targeting NO to the NMDAR nitrosylation site; (ii) hypoxia enhances downregulation of NMDAR activity via S-nitrosylation; (in) a novel family of NMDAR subunits, NR3A and NR3B, exist in the brain (we have cloned and characterized these subunits). Co-expression of NR1/NR2A/NR3A(3B) in recombinant systems decreases unitary conductance, Ca2+ permeability, and Mg2+ sensitivity in single-channel recordings of NMDA/glycine-activated currents. Decreased NMDAR activity engendered by NR3 may be protective during normal development; (iv) co-expression of NR3 and NR1 subunits (without NR2) in Xenopus oocytes and mammalian HEK 293 cells form excitatory lycine-activated cation channels (in the absence of glutamate); (v) S-nitrosylation of the NR1 structure in crystallography experiments. This finding allows us to begin to relate structure to function. We propose the following Specific Aims: [1] To prove the specificity of NO-Memantine targeting to the NMDAR, and to elucidate the mechanism of enhanced downregulation of NMDAR activity by S-nitrosylation under hypoxic conditions. Importantly, drugs studied here in Project I will be used for neuroprotection experiments in the other Projects of this P01 grant; [2] To characterize the channel pore region of NR3-containing channels; [3] To characterize the ligand-binding site of NR3-containing receptors, and use information from [2] and [3] to distinguish expression of NR1/NR3 "doublet" receptors from NR1/NR2/NR3 "triplet" receptors. [4] To further study the structure of NMDAR subunit ligand-binding and nitrosylation domains using crystallography.